The predecessor grant for this competing continuation application proposed several lines of investigation to understand the function of the novel channel/kinase protein TRPM7 (previously designated LTRPC7), and work funded under that application defined a key role for TRPM7 in regulating and/or mediating vertebrate cellular Mg2+ uptake required for cell proliferation. This competing continuation has been broadened beyond studies focused on TRPM7 to address a range of questions involving TRPM7 function and its relationship to mechanisms which regulate cellular Mg2+ homeostasis and cell growth. In Specific Aim 1, the integration of TRPM7's Mg2+ uptake capacity with the regulation of cell growth and proliferation will be analyzed, with a focus on understanding how access to Mg2+ influences nutrient sensing translational control pathways. Specific Aim 2 proposes studies aimed at identifying molecular and functional targets of a hypothesized signaling function of the TRPM7 kinase domain. Subaim 2a) will use in vitro studies to define a preferred substrate motif and generate phosphospecific antibody reagents as an approach to identifying direct molecular targets of the kinase domain, while subaim 2b) will create and characterize "analog sensitive" TRPM7 mutants for use in molecular and in-vivo approaches to identifying targets of TRPM7 kinase domain signaling function. Specific Aim 3 will focus on characterizing the role of a novel Mg2+ transporter, designated SLC41A2, in DT40 cell Mg2+ homeostasis through the creation and characterization of an SLC41A2-deficient DT40 cell line. Together, the proposed studies will generate a foundation of knowledge on the molecular mechanisms of Mg uptake and their integration with mechanisms which regulate cell growth and proliferation.